JP6698358B2 - A sweat secretagogue and a preventive or therapeutic agent for dry skin containing the sweat secretagogue - Google Patents

Description

  The present invention relates to a sweat secretagogue and a preventive or therapeutic drug for dry skin containing the sweat secretagogue.

  Dry skin is a state in which the skin is dry because the barrier function of the skin is lowered. Further, since the barrier function of the skin is lowered, it is likely to be directly exposed to external stimuli, and it is likely to cause symptoms such as rough skin and itchy skin. The main causes of dry skin include atopic dermatitis, aging, and dryness in winter.

  As a therapeutic agent for dry skin, for example, an oral skin moisturizer containing an extract of the family Brassicaceae as an active ingredient (Patent Document 1), a skin roughening preventive agent and a mucous membrane repair agent characterized by containing pectin as an active ingredient ( Patent Document 2), a bath preparation composition containing a hyperthermia medicinal plant or its extract, urea and vitamin A (Patent Document 3) and the like have been reported, but further development of a new therapeutic drug for dry skin is required. Be done.

In addition, many of the therapeutic agents for dry skin are aimed at preventing the dryness of the skin by covering the surface of the skin and alleviating the symptoms of dry skin. No therapeutic agent has been known so far.
Therefore, development of a therapeutic agent for dry skin that promotes sweat secretion by activating the molecular mechanism is desired.

  On the other hand, PACAP (Pituitary Adenylate Cycle-Activating Polypeptide) is a kind of neuropeptide and is a peptide consisting of 27 or 38 amino acid residues. The use of PACAP as a neurite inducer, an anti-inflammatory agent, a chronic lung disease therapeutic agent, and an eye disease therapeutic agent has been reported so far (Patent Documents 4 to 8 and Non-Patent Document 1). However, it has not been reported that PACAP is involved in promoting sweat secretion.

JP, 2005-281127, A JP, 2004-059440, A JP, 09-002939, A JP 2001-226284 A JP, 2004-224775, A JP, 2004-315436, A JP, 2006-306770, A JP, 2009-269818, A

Vaudry D, Falluel-morel A, Bourgault S, Basille M, Bure D et al., Pituitary Adenylate Cyclase-Activating Polypeptide and Its Receptors: 20 years after the Discovery. Pharmacol Rev 2009;61(3);283-357.

  The present invention has been made in view of the above background art, and an object thereof is to provide a novel preventive or therapeutic drug for dry skin.

  As a result of intensive studies to solve the above problems, the present inventors have found that PACAP is involved in promoting sweat secretion, and have completed the present invention.

  That is, the present invention provides a sweat secretagogue, which comprises PACAP or a pharmaceutically acceptable salt thereof as an active ingredient.

  The present invention also provides a preventive or therapeutic agent for dry skin, which comprises the above sweat secretagogue.

  ADVANTAGE OF THE INVENTION According to this invention, the said problem and the said subject can be solved, and a sweat secretion promoter which can be used for prevention or treatment of dry skin can be provided.

  Further, the preventive or therapeutic agent for dry skin of the present invention is different from the conventional preventive or therapeutic agent for dry skin, which aims to retain water (moisturizing) of the skin, for the purpose of hydrating the skin by promoting sweat secretion. And is extremely effective for the prevention or treatment of dry skin.

It is a figure which shows the result of PAC1R immunopositive reaction in a mouse plantar (upper) and human plantar (lower). It is the figure which showed the PAC1R immunity positive reaction in the mouse foot sole. The localization of PAC1R (lower left), the localization of SMA (lower right), and the co-localization of PAC1R/SMA/DAP1 (upper right) were confirmed. It is a figure of the photograph which showed the PAC1R immunopositive reaction in the human plantar. The localization of PAC1R (lower left), the localization of SMA (lower right), and the co-localization of PAC1R/SMA/DAP1 (upper right) were confirmed. It is the figure which compared the expression level of the mRNA of PACAP receptor (PAC1R, VPAC1R, VPAC2R), VIP, and PACAP in mouse skin using RT-PCR. It is the figure which compared the expression level of PACAP receptor (PAC1R, VPAC1R, VPAC2R), VIP, and PACAP mRNA in the tissue extract obtained from the mouse planta using RT-PCR. It is a protocol used when evaluating the sweat secretion promoting effect by PACAP administration. Physiological saline (Saline) (top) or PACAP (10 ⁻⁶ M) (bottom) was locally administered to the plantar sole of the mouse before local administration (left) and 120 minutes after the local administration (right). ) Is a photograph comparing the amount of perspiration. It is the graph which showed the dosage change and the time change of the sweat secretion amount after PACAP administration. (A) is a graph showing changes in the amount of sweat secretion after administration of PACAP and changes over time. (B) It is a graph which shows the verification result of whether the amount of sweat secretion after PACAP administration is affected by anesthesia. (C) It is a graph which shows the verification result of whether the sweat secretion promotion effect by PACAP is local. It is a protocol used when evaluating the receptor involved in the sweat secretagogue effect by PACAP administration. (A) It is the figure of the photograph which compared the perspiration amount 120 minutes after starting an experiment by the protocol described in (i)-(iv) of FIG. 10 ((i) control, (ii) experiment start 10 Minutes later, PACAP was administered, (iii) PACAP6-38 was administered immediately after the start of the experiment, PACAP was administered 10 minutes after, and (IV) VIP was administered 10 minutes after the start of the experiment). (B) A graph showing changes in sweat secretion and time after administration of PACAP, PACAP and PACAP6-38, or VIP. It is the figure which compared the expression level of the mRNA of PACAP and PAC1R in the skin of the human plantar using RT-PCR.

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments and can be arbitrarily modified within the scope of the technical idea.

[Sweat secretagogue]
The sweat secretagogue of the present invention is characterized by containing PACAP or a pharmaceutically acceptable salt thereof as an active ingredient.

PACAP (Pituitary Adenylate Cycle-Activating Polypeptide) is a neuropeptide consisting of 27 or 38 amino acid residues that is strongly expressed in the central and peripheral nervous systems and is highly expressed in the testis, adrenal gland, intestinal tract and the like. Widely distributed in peripheral tissues.
VCAP receptors (VPAC1 receptor, VPAC2 receptor) that bind to the receptor of PACAP with the same affinity to VIP (Vasoactive intestinal polypeptide: vasoactive intestinal polypeptide) and accelerate the production of cAMP And PAC1 receptor that selectively binds to PACAP and activates phosphatidylinositol turnover and MAP kinase in addition to cAMP production.

  The present invention was made for the first time by finding that PACAP promotes sweat secretion, as shown in Examples and the like.

  Further, the sweat secretagogue of the present invention preferably contains one or more peptides belonging to the "group consisting of PACAP and a pharmaceutically acceptable salt thereof".

  The method for synthesizing PACAP used in the present invention is not particularly limited, but it can be synthesized according to a known peptide synthesis method. Examples thereof include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carbimidazole method, a redox method, and a DCC-additive method. These synthetic methods can be applied to both solid phase synthesis and liquid phase synthesis.

  Examples of pharmaceutically acceptable salts of PACAP include salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; aluminum salts; and inorganic bases such as ammonium salts. Salts; salts with organic bases such as trimethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N-dibenzylethylenediamine; hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with inorganic acids, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. And a salt thereof with a polymeric acid such as tannic acid, carboxymethyl cellulose, polylactic acid, and polyglycolic acid;

Furthermore, the sweat secretagogue of the present invention may contain a PACAP derivative or a pharmaceutically acceptable salt thereof.
The PACAP derivative is, for example, one in which a part of amino acids in the polypeptide structure of PACAP has been deleted or substituted, or one in which another amino acid has been inserted in the polypeptide structure of PACAP and which has a sweat secretagogue action. To tell.

As the pharmaceutically acceptable salt of the PACAP derivative, the synthetic method and the like, the same ones as those of PACAP described above can be used or applied.
The PACAP or PACAP derivative or a salt thereof is preferably isolated or purified, and preferably extracted or synthesized.

The content of PACAP or PACAP derivative or a pharmaceutically acceptable salt thereof in the sweat secretagogue is not particularly limited as long as it can promote sweat secretion, and can be appropriately selected according to the purpose. The total amount of PACAP or PACAP derivative or a pharmaceutically acceptable salt thereof is preferably 10 ⁻¹² mol/L to 10 ⁻⁴ mol/L with respect to the entire sweat secretagogue, The content is more preferably 10 ⁻¹¹ mol/L to 10 ⁻⁵ mol/L, and particularly preferably 10 ⁻¹⁰ mol/L to ^{10 −6} mol/L.

  Any one of the above-mentioned PACAP, PACAP derivative, and pharmaceutically acceptable salt thereof may be contained in the sweat secretagogue, or two or more thereof may be used in combination. When two or more kinds are used in combination, the content ratio of each compound in the sweat secretagogue is not particularly limited and can be appropriately selected according to the purpose.

  In addition, the sweat secretagogue of the present invention may contain "other components" in addition to PACAP, PACAP derivatives, and pharmaceutically acceptable salts thereof.

The above-mentioned "other components" in the sweat secretagogue are not particularly limited and may be appropriately selected depending on the purpose within a range not impairing the effects of the present invention, and for example, pharmaceutically acceptable. The carrier etc. which can be obtained are mentioned.
The carrier is not particularly limited and can be appropriately selected depending on, for example, the dosage form described below. The content of the "other components" in the sweat secretagogue is also not particularly limited and may be appropriately selected depending on the purpose.

[Preventive or therapeutic drug for dry skin]
The preventive or therapeutic drug for dry skin of the present invention contains the above-mentioned sweat secretagogue.

  The content of the sweat secretagogue with respect to the entire dry skin preventive or therapeutic agent of the present invention is not particularly limited and can be appropriately selected according to the purpose, but 100 parts by mass of the whole natural immunity activator. In this case, the sweat secretagogue is preferably 0.001 to 100 parts by mass, more preferably 0.01 to 99 parts by mass, particularly preferably 0.1 to 95 parts by mass, and further preferably 1 ˜90 parts by mass.

The dosage form of the dry skin preventive or therapeutic agent of the present invention is not particularly limited and can be appropriately selected depending on, for example, a desired administration method as described below.
Specifically, for example, oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), oral liquid preparations (internal solution, syrup, elixir, etc.), injections (solvents, suspensions, etc.) , Ointments, patches, gels, creams, external powders, sprays, inhalation sprays and the like.

As the oral solid agent, for example, an excipient, and if necessary, an additive such as a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent and a flavoring agent is added to the sweat secretagogue. Can be manufactured by a conventional method.
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid and the like.
Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. Be done.
Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose and the like.
Examples of the lubricant include refined talc, stearate, borax, polyethylene glycol and the like.
Examples of the colorant include titanium oxide and iron oxide.
Examples of the corrigent/flavoring agent include sucrose, orange peel, citric acid, tartaric acid, and the like.

The oral liquid preparation can be produced by a conventional method, for example, by adding additives such as a corrigent/flavoring agent, a buffer and a stabilizer to the sweat secretagogue.
Examples of the corrigent/flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate and the like. Examples of the stabilizer include tragacanth, gum arabic, gelatin and the like.

As the injection, for example, a pH regulator, a buffer, a stabilizer, a tonicity agent, a local anesthetic and the like are added to the sweat secretagogue, and subcutaneous, intramuscular, and intravenous injections are carried out by a conventional method. It is possible to produce an injection for internal use and the like.
Examples of the pH adjuster and the buffer include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid and the like. Examples of the tonicity agent include sodium chloride, glucose and the like. Examples of the local anesthetic include procaine hydrochloride, lidocaine hydrochloride and the like.

The ointment can be produced, for example, by adding a known base, stabilizer, humectant, preservative and the like to the above-mentioned sweat secretagogue and mixing them by a conventional method.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, paraffin and the like. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like.

  The patch can be produced, for example, by applying a cream, gel, paste or the like as the ointment to a known support by a conventional method. Examples of the support include woven fabrics, non-woven fabrics, soft vinyl chloride, polyethylene, polyurethane films made of cotton, staple fibers, and chemical fibers, foam sheets, and the like.

  The preventive or therapeutic agent for dry skin of the present invention is, for example, an individual in need of activation of a sweat secretion promoting mechanism (for example, an individual in need of health maintenance or sweat secretion; prevention or treatment of cancer or lifestyle-related diseases). It can be used by administering to an individual in need thereof; an individual infected with bacteria, fungi, viruses and the like).

  The animals to which the preventive or therapeutic agent for dry skin of the present invention is administered are not particularly limited, and examples thereof include humans; mice; rats; monkeys; horses; livestock such as cows, pigs, goats, chickens; cats, dogs, etc. Pets; and the like.

The method for administering the dry skin preventive or therapeutic agent is not particularly limited, and can be appropriately selected depending on the dosage form of the sweat secretagogue, for example, oral administration, intraperitoneal administration, blood Injection into the intestine, infusion into the intestine and the like.
In addition, the dose of the dry skin preventive or therapeutic agent is not particularly limited and may be appropriately selected depending on the age, weight, desired degree of effect, etc. of the individual to be administered. The daily dose to an adult is preferably 1 mg to 30 g, more preferably 10 mg to 10 g, and particularly preferably 100 mg to 3 g as the amount of the active ingredient.
Further, the timing of administration of the dry skin preventive or therapeutic agent is not particularly limited and may be appropriately selected according to the purpose. For example, it may be administered prophylactically or therapeutically. Good.

  Hereinafter, the present invention will be described in more detail based on Examples and Test Examples, but the present invention is not limited to the specific ranges of Examples and the like below. "%" indicates "mass %" unless otherwise specified.

[Experimental animals]
Mice (wild type C57BL/6J, male) were purchased from Sankyo Lab Service Co., Ltd., and male mice aged 15 to 19 weeks were used. All experimental methods related to animal experiments have been approved by the Animal Experiment Committee of Showa University.

[Human sample used in the experiment]
Human skin samples (mean age 45.3±14 years, 2 males, 4 females) were obtained from patients who underwent surgery to remove benign tumors of the sole of the foot. All patients gave informed consent and consented to participate in this experiment. The normal site of the excised skin was used for the experiment. This experiment has been approved by the Ethics Committee of Showa University School of Medicine.

[Total RNA extraction and RT-PCR]
Known techniques were used for total RNA extraction and RT-PCR. Total RNA was extracted from the sample powder using QIAGEN RNeasy Mini Kit (QIAGEN). First, the total RNA sample was treated with RNase-free DNase (Stratagene), and cDNA was synthesized in 20 μL of the reaction mixture using AffinityScript QPCR cDNA Synthesis Kit (Stratagene). RT-PCR was performed in a reaction mixture containing cDNA, and a primer set, Emerald Amp PCR Master Mix (Takara Shuzo), and S1000 thermal cycler (BIO RAD) were used. Then, electrophoresis was performed at 100 V for 25 minutes in a 1% TAE buffer. The gel was stained with ethidium bromide, and the stained band was visualized using ChemiDoc XRS+ (BIO RAD).

[Evaluation of sweat secretion by Wada-Takaki method]
The sweat glands that secreted sweat were visualized using the Wada-Takaki method (a modified method of the Miller method utilizing the iodine starch reaction). C57BL/6J mice (n=10) were anesthetized with 10 μL of pentobarbital (Kyoritsu Pharmaceutical Co., Ltd.) per 1 g of body weight, and then 10% povidone-iodine solution (Hauzou Medical Co., Ltd.) was applied to the paws of the mice. After drying, it was coated with castor oil containing 50% corn starch liquid (Wako Pure Chemical Industries, Ltd.). 5 μL of PACAP (concentration: 0, 1×10 ⁻¹⁰ , 1×10 ⁻⁸ , 1×10 ⁻⁶ (mol/L)) was subcutaneously injected into the toes based on the protocol shown in FIGS. 6a and 6b. In another experiment, saline or 1×10 ⁻⁵ mol/L PACAP6-38 was injected 10 minutes before administration of 1×10 ⁻⁶ mol/L PACAP or VIP (FIG. 10). The black dots in the photograph indicate that they are sweating, and quantitative analysis was performed by counting the number of black dots.

[Immunohistochemistry]
Mice were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally), perfused transcardially with physiological saline, and then perfused with 50 mM phosphate buffer (pH 7.2) containing 4% paraformaldehyde (PFA). did. The toes were collected, 4% PFA was added and left at 4°C overnight. After immersing in 0.1 M phosphate buffer (pH 7.2) containing 20% sucrose at 4° C., the toes were placed on the O.D. C. T. It was embedded in a compound (Sakura Finetech Japan Co., Ltd.) and frozen. Frozen sections were microtone cut to a thickness of 5 μm.
Human tissues used in the experiment were O.I. C. T. It was embedded in a compound (Sakura Finetech Japan Co., Ltd.) and frozen with liquid nitrogen. Frozen sections were microtone cut to a thickness of 5 μm. To human samples, 4% PFA was added 30 minutes before immunostaining.

[[Single immunostaining]]
Microscope slides containing tissue sections were washed with phosphate buffered saline (PBS, pH 7.2). The sections were treated under citric acid (10 mM, pH 6.0) at 85°C for 20 minutes. After treatment with PBS containing 0.3% hydrogen peroxide to stop the reaction of endogenous peroxidase, sections were treated with PBS containing 5% normal horse serum (NHS) for 60 minutes to prevent nonspecific binding. .. The sections were then incubated overnight at 4° C. in the presence of rabbit anti-PAC1R polyclonal antibody (1:400, made by the inventors). Then, the section was washed with PBS, incubated in the presence of biotinylated goat anti-rabbit IgG (1:200, Santa Cruz Biotechnology) at room temperature for 2 hours, and reacted with an avidin-biotin complex solution (Vector). Then, diaminobenzidine (Vector) was used as a chromogen. Nuclei were counterstained with hematoxylin. Single immunostaining was detected using a microscope (AX70, Olympus).

[[Double immunostaining]]
The sections were washed with PBS (pH 7.2) and treated under citric acid (10 mM, pH 6.0) at 85°C for 20 minutes. Thereafter, the sections were treated with PBS containing 5% normal horse serum (NHS) for 60 minutes. Then, the sections were overnight in the presence of a rabbit anti-PAC1R polyclonal antibody (1:400, prepared by the present inventors) as a primary antibody and a mouse anti-smooth muscle actin (SMA) monoclonal antibody (1:400, R&D SYSTEMS). Incubated. The sections were then washed with PBS and visualized with Alexa546 anti-rabbit IgG antibody (1:400, Life Technologies) and AlexaFluor488 anti-mouse IgG antibody (1:400, Life Technologies). The nuclei were then stained with 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride (1:10000, Roche) for counterstaining. Images were acquired using ApoTome (Zeiss, FIG. 2) and Nikon A1 confocal microscope (Nikon, FIG. 3).

[Statistical analysis]
Data are shown as mean±SEM. Experimental results were evaluated using the Tukey-Kramer HSD test. When p<0.05, it was judged to be statistically significant.

Test example 1
<Comparison of expression levels in sweat glands>
In humans, the sweat glands are roughly divided into eccrine sweat glands and apocrine sweat glands, whereas only eccrine sweat glands are present in mice. The sweat gland is composed of an acinar portion, myoepithelial cells covering the acinar portion, and a conduit portion. Sweat is produced in the acinar portion, and the sweat is secreted as sweat through the conduit portion. In addition, sweat secretion is regulated by the sympathetic nerve.

  First, fluorescent immunostaining was used to examine the localization of PACAP receptors, and 7 μm frozen sections of mouse foot soles and 5 μm frozen sections of human foot soles were used, which are one of the PACAP receptors as primary antibodies. PAC1 receptor antibody (produced by the present inventors), SMA antibody (R&D SYSTEMS, Minneapolis, USA, 1:400) which is a myoepithelial cell marker, Alexa546 labeled anti-rabbit IgG (life technologies, Carlsbad, CA, 1:400), Alexa488-labeled anti-mouse IgG (life technologies, 1:400), and fluorescent confocal microscope A1 (Nikon, Tokyo, Japan) after nuclear staining with DAPI (Roche, Mannheim, Germany, 1:10000). ) Was used to observe the positive reaction of the PAC1 receptor. The results are shown in FIGS.

  According to the RT-PCR (Reverse Transcription Polymerase Chain Reaction) method, RNA was extracted from mouse skin and cDNA was prepared by reverse transcription reaction. Primers for PACAP, VIP, PAC1 receptor (sometimes abbreviated as "PAC1R"), VPAC1 receptor (sometimes abbreviated as "VPAC1R"), VPAC2 receptor (sometimes abbreviated as "VPAC2R") Was used to perform PCR. The results are shown in Fig. 4.

From the observation results of fluorescent immunostaining, PAC1R positive cells were found in the sweat glands (FIGS. 1 to 3). Double-staining of a sweat gland section of a mouse with PAC1R (red) and smooth muscle actin (SMA, green) revealed many PAC1R-positive cells in secretory cells, but SMA and PAC1R staining did not overlap. (Figure 2).
In addition, immunohistochemical staining revealed that the localization of PACAP and PAC1R in the sweat glands of human and mouse soles was similar. By immunohistological staining, strong staining of PAC1R was observed in eccrine gland secretory cells, staining was also observed in ducts, and similar results were obtained in mice (FIG. 1). As a result of double-staining human sweat glands with PAC1R (red) and SMA (green), many PAC1R-positive cells were confirmed in secretory cells (FIG. 3).
These results suggest that the role of PACAP in sweat secretion is similar in humans and rodents.

  By RT-PCR, expression of PACAP, VIP, PAC1R, VPAC1R, and VPAC2R mRNA was observed in mouse skin (FIG. 4).

  Furthermore, it was examined by RT-PCR whether or not PACAP and its receptor were expressed in the tissue extract obtained from the sole of the mouse. PACAP, VIP, PAC1R, VPAC1R, and VPAC2R mRNA transcripts were detected in toes skin (FIG. 5). The mRNA expression levels of VIP and PAC1R were high, whereas the mRNA expression levels of PACAP, VPAC1R and VPAC2R were lower than those of VIP and PAC1R.

Test example 2
<Promotion of sweat secretion by PACAP>
Next, physiological saline (saline, vehicle), PACAP38 (Peptide Institute, Osaka) (hereinafter sometimes simply abbreviated as "PACAP") was subjected to intraperitoneal anesthesia under pentobaltar (10) according to the protocol shown in FIG. 6a. Double dilution, 10 μL/body weight 1 g) was added to the plantar (C57BL/6J, male, 18 to 20 weeks old) of the mouse, and the concentration was changed from 10 ⁻¹⁰ to 10 ⁻⁶ M (mol/L), and the body weight was 1 g. A local injection of a volume of 5 μL per injection was performed. The expression level of sweat was measured by the Wada-Takaki method (modified Minor method).

Specifically, 10% povidone-iodine solution was applied to the plantars before local injection of PACAP, and after sufficiently dried, a mixture of soluble starch and castor oil was thinly applied, and iodine was made to coincide with the pores of the sweat with the start of sweating. A starch reaction occurs, and minute black colored points appear.
With this, the soles were photographed with a digital camera before, 60 minutes, 90 minutes, and 120 minutes after administration of PACAP, and the number of black spots was counted and measured. The higher the number of perspiration black points, the more the sweat secretion was promoted.

FIG. 7 shows the results of photographing black spots of sweating before administration of physiological saline and PACAP (10 ⁻⁶ M) and at 120 minutes after administration. FIG. 7 (top) shows the case where physiological saline (Saline) was administered, and FIG. 7 (bottom) shows the case where PACAP was administered. The arrow in FIG. 7 indicates a part of the black dot of sweating.
Compared to saline administered, the PACAP10 ^-6 M dose, sweating was observed after 120 minutes.

The changes in sweat secretion after PACAP administration are shown in FIGS. 8 and 9a by counting the number of black spots before PACAP administration and 60 minutes, 90 minutes, and 120 minutes after administration.
In PACAP10 ^-6 M dose group compared to the physiological saline-administered group (saline or vehicle), the amount of perspiration was significantly increased after 120 minutes (Figure 8, Figure 9a). On the other hand, when PACAP10 ^-10 M or PACAP10 ^-8 M was administered, a statistically significant increase in sweating was not observed. Sweat secretion by the PACAP effect was promoted in a dose-dependent manner.

When pilocarpine (a non-selective muscarinic receptor agonist) was injected into the mouse toes, functional sweet gland was observed after 15 minutes (not shown). On the other hand, in the case of PACAP, sweating was confirmed after 120 minutes.
Therefore, in order to confirm whether or not the effect of PACAP was affected by anesthesia, PACAP or a control was injected into the mouse toe pad 60 minutes after anesthesia according to the protocol shown in FIG. 6b.

The results are shown in Figure 9b. In mice injected with 1×10 ⁻⁶ mol/L (M) of PACAP, sweat secretion was not increased 60 minutes after injection of PACAP (120 minutes after anesthesia) (FIG. 9b). (Left), the amount of sweat secretion greatly increased 90 minutes after injection of PACAP (150 minutes after anesthesia) (Fig. 9b right). This result suggested that the effect of sweat secretion by PACAP was slow, and the effect began to appear when anesthesia was cut off.

Further, in order to verify whether the effect of PACAP is local or exerts an effect on the whole, the difference in the amount of sweat secretion between the side to which PACAP was administered and the side not to be administered was examined.
The verification result is shown in FIG. 9c. The amount of sweat secretion was significantly increased only on the side injected with PACAP (injected side), and the amount of sweat secretion was not increased on the non-injected side of the foot on the opposite side (Fig. 9c). This result suggests that PACAP promoted the secretion of sweat by locally reacting with the receptor present in the mouse toes. More surprisingly, normal sweat secretion was confirmed in PACAP knockout mice after pilocarpine administration (not shown).
These results suggest that PACAP is involved in sweat secretion via an acetylcholine receptor-dependent pathway.

Test example 3
<Search for receptors involved in sweat secretion promotion by PACAP>
Next, in order to investigate which receptor is involved in the effect by PACAP, verification was performed using an antagonist of PAC1R (PACAP6-38) and an antagonist of VPAC1R and VPAC2R (VIP) according to the protocol shown in FIG. 10. It was

The results are shown in Fig. 11. (Ii) When only 1×10 ⁻⁶ mol/L of PACAP was injected, the amount of sweat secretion greatly increased 120 and 150 minutes after the start of the experiment. On the other hand, (iii) when PACAP was administered immediately after the start of the experiment, PACAP 6-38, and 10 minutes later, the amount of sweat secretion did not significantly increase. (Iv) Even when VIP was administered 10 minutes after the start of the experiment, the amount of sweat secretion did not significantly increase.
From these results, it was suggested that topical administration of PACAP promotes sweat secretion, and its effect is exerted via PAC1R expressed in sweat glands.

Test example 4
<Expression of PACAP and PAC1R in human>
RT-PCT was performed in order to demonstrate the difference in the expression levels of PACAP and PAC1R mRNA in human foot skin between individuals. The expression level of PACAP mRNA was significantly lower in plantar dermis than in the brain (Fig. 12). On the other hand, the expression level of PAC1R was not significantly different between the skin of the sole and the brain. Comparing the two human samples, there was no difference in the expression levels of PACAP and PAC1R. The above results suggest that there was no significant difference in the expression level among human individuals.

<Discussion>
This test example proved the role of PACAP in the sweat glands, which promotes the secretion of sweat in vivo. It was confirmed that PACAP and its receptor were expressed in mouse skin, and it was found that administration of PACAP induces sweat secretion via PAC1R. Most of the PAC1R positive cells were observed in secretory cells of mouse and human sweat glands. These results indicate that PACAP plays an important role in sweating, and also indicate that understanding the pathophysiology of sweating disorders is important. Then, it was shown for the first time that the sweat secretion by PACAP in the eccrine gland was promoted by this test example and the like.

The effect of PACAP on sweat secretion was verified, and the secretion of sweat was promoted in a wild-type mouse subcutaneously injected with PACAP in a dose-dependent manner. Although it is already known, sweating is promoted by acetylcholine stimulation via muscarinic receptors. When pilocarpine was administered to the toes of mice, a functional sweat gland was detected at 5 minutes (not shown). On the other hand, when PACAP was administered, it was observed after 120 minutes (Fig. 9d). This data suggests that PACAP indirectly stimulates muscarinic receptors or is involved in other sweating pathways or mechanisms in the sweat glands.
Previous studies have reported that aquaporin-5 (AQP5) is involved in secretion in salivary glands, submucosa and sweat glands. AQP5 is rapidly and instantaneously phosphorylated by PKA via the cAMP signaling pathway in the apical membrane of acinar cells of the submandibular and parotid glands after administration of isoproterenol. On the other hand, pilocarpine administration does not result in phosphorylation. Therefore, it is considered that AQP5 may be involved in sweat secretion by PACAP.

  Intravenous injection of PACAP in vivo in healthy humans has been reported to induce vasodilation, flushing and edema via VPAC1R after 15 minutes (peak after 30 minutes). In addition, it was reported that, when PACAP was intravenously injected into rats, promotion of salivary secretion was observed in three main salivary glands. Considering the results of the present invention, it is suggested that PACAP has an important role in various exocrine secretion and vasodilation in the skin. The fact that PACAP exhibits contradictory functions strongly suggests that the action of PACAP is greatly influenced by the receptor subtype, and that other nutrition-related factors and signal transduction molecules are present.

  Previous studies on the eccrine gland suggest that VIP stimulates sweat secretion by increasing cAMP levels and acts as a synergist for both acetylcholine- and agonist-mediated sweat secretion. ing. On the other hand, PACAP administration changed the intracellular localization of AC in human sweat glands, showing a much stronger change than VIP stimulating the secretion of adenylate cyclase, cAMP and catecholamines. The present inventors demonstrated that sweat administration was not promoted by VIP administration (FIG. 11). PACAP may play a more important role than VIP in eccrine glands via cAMP.

  The localization of PACAP receptors in the sweat glands and their role in sweat secretion have not been previously demonstrated. According to the present invention, it was found that most of the PAC1R positive cells are present in the secretory cells of mouse and human sweat glands (FIG. 1). Sweat secretion requires contraction of myoepithelial cells that surround the secretory cells, but PAC1R-positive cells are not important in myoepithelial cells. This data suggests that PACAP is not involved in promoting the secretion of sweat by myoepithelial cells surrounding the secretory cells, but promoting the secretory activity itself of the secretory cells.

  In summary, it is considered that local administration of PACAP promotes sweat secretion, and its effect is mediated by PAC1R expressed in secretory cells of sweat glands. It is necessary to examine whether PACAP promotes sweat secretion in healthy people and patients with clinical disorders. PACAP and its receptors may offer new therapeutic approaches and may provide insights into new mechanisms of sweating in the presence of clinical disorders.

<Summary of Examples>
According to the present invention (particularly from Test Example 2), it was found that PACAP promotes sweat secretion.
According to the present invention (particularly from Test Example 1), it was revealed that PACAP induces sweat secretion promotion by activating any PACAP receptor, and Test Example 3 induces sweat secretion promotion via PAC1R. It was suggested to do. It was also confirmed that the sweat glands have a PACAP receptor. Furthermore, it was suggested that pharmaceutically acceptable salts of PACAP, PACAP derivatives or pharmaceutically acceptable salts thereof may induce sweat secretion promotion by activating the PACAP receptor.

  The sweat secretagogue of the present invention can be used not only as a medicine for improving symptoms of dry skin, but also as a cosmetic for dry skin care, a functional food and the like.

Claims (2)

  What is claimed is: 1. A PAC1 receptor stimulator having a pituitary adenylate cyclase activating polypeptide (PACAP) localized in secretory cells of a sweat gland, which is expressed in the eccrine sweat gland of a subject by topical administration to the subject. A PAC1 receptor stimulant for stimulating a receptor for promoting secretion of sweat in a subject. A sweat secretagogue comprising the PAC1 receptor stimulant according to claim 1.